EP2928907A1 - Peptides pénétrant dans les cellules pour la livraison intracellulaire de molécules - Google Patents

Peptides pénétrant dans les cellules pour la livraison intracellulaire de molécules

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Publication number
EP2928907A1
EP2928907A1 EP13773244.2A EP13773244A EP2928907A1 EP 2928907 A1 EP2928907 A1 EP 2928907A1 EP 13773244 A EP13773244 A EP 13773244A EP 2928907 A1 EP2928907 A1 EP 2928907A1
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Prior art keywords
cell
vepep
complex
peptide
peptides
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EP13773244.2A
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German (de)
English (en)
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EP2928907B1 (fr
Inventor
Gilles Divita
Sébastien DESHAYES
Karidia KONATE
May Catherine Morris
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Aadigen LLC
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Centre National de la Recherche Scientifique CNRS
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin

Definitions

  • the present invention pertains to the field of intracellular delivery of molecules such as nucleic acids and small hydrophobic molecules.
  • the invention relates to a new cell-penetrating peptide (CPP) family, which exhibits a high efficacy and a low toxicity.
  • CPP cell-penetrating peptide
  • CPP Cell Penetrating Peptides
  • CPPs from both strategies have been reported to favour the delivery of a large panel of cargos (plasmid DNA, oligonucleotide, siRNA, PNA, protein, peptide, liposome, nanoparticle%) into a wide variety of cell types and in vivo models [3-7].
  • VEPEP-5 10 amino acid cell- penetrating peptides for the delivery of peptides/proteins and hydrophobic molecules. Delivery strategies using VEPEP-5 peptides as the outer layer of nanoparticles are referred to as NANOPEP-5.
  • VEPEP-5 are short primary and, in certain cases, secondary amphipathic 10 amino acid peptides forming stable nanoparticles with molecules such as peptides, peptide-analogues, PNAs and small hydrophobic molecules, hereafter designated as "SHM”.
  • VEPEP-5 vectors comprise or consist of an amino acid sequence consisting of a beta-alanine or a serine in N-terminal position, linked to a sequence consisting of 9 consecutive amino acids from the sequence RXWXRLWXRLR (SEQ ID NO: 7), wherein:
  • X in position 2 is R or S; and X in positions 4 and 8 arc, independently from each other. W or F.
  • Non-limitative examples of VEPEP-5 peptides according to the present invention comprise an amino acid sequence selected from the group consisting of:
  • is beta- A or S.
  • the present invention also pertains to a stapled cell-penetrating peptide derived from a VEPEP-5 cell-penetrating peptide as described above.
  • a "stapled" peptide designates a peptide which comprises a chemical linkage (in addition to the amino acid chain) between two residues.
  • the VEPEP-5 peptide comprises a hydrocarbon linkage between two residues which arc separated by three or six residues.
  • the skilled artisan can obtain these peptides by using techniques which are available in the art, for example as described by Verdi ne and Hilinski, Methods in Enzymology, 2012 [12].
  • VEPEP-5 strategy improves both ex-vivo and in vivo delivery and efficiency of peptide/protein/peptide analogue and small hydrophobic molecules, without activating the innate immune response or inducing toxic side effects.
  • a cell-penetrating peptide of the present invention further comprises, covalently linked to the N-terminal end of the amino acid sequence, one or several chemical entities selected in the group consisting of an acetyl, a fatty acid, a cholesterol, a poly-cthylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide and a targeting molecule (peptide, fatty acid, saccharide).
  • VEPEP-5 peptide is advantageous for stabilizing nanoparticles in vivo.
  • a cell-penetrating peptide according to the invention can comprise, covalently linked to the C-terminal end of its amino acid sequence, one or several groups selected in the group consisting of a cysteamide, a cysteine, a thiol, an amide, a nitrilotriacetic acid optionally substituted, a carboxyl, a linear or ramified C1-C6 alkyl optionally substituted, a primary or secondary amine, an osidic derivative, a lipid, a phospholipid, a fatty acid, a cholesterol, a. poly-ethylene glycol, a nuclear localization signal, nuclear export signal, an antibody, a polysaccharide and a targeting molecule.
  • Another aspect of the present invention is a complex comprising a cell- penetrating peptide as described above and a cargo selected amongst protein/peptide and hydrophobic molecules.
  • polypeptide cargoes are proteins of at most 15kDa, such as nanobodies, small peptides, cyclic peptides, peptide-based biomarkers, bio-drugs, PNAs or oligonucleotides.
  • the cargo is a small molecule (size lower than 1.5 kDa), either hydrophobic or charged.
  • Preferred cargos in the complexes according to the present invention are anticancer and antiviral drugs, as well as cosmetic agents.
  • Non- limitative examples of small hydrophobic molecules which can be used include amino acids, di- or tri- peptides (labelled or not) daunomycin, Paclitaxel, doxorubicin, AZT, porphyrin, fluorescently- labelled-nucleosides or nucleotides (FA -Guanosine, CY5_UTP, CY3-UTP), hydrophobic maghemite (contrast agents or magnetic nanoparticles Fe 2 0 3 ) and fluorescent dyes.
  • the size of the complexes described above is preferably between 50 and 200 nm (the size of the complex herein designates its mean diameter).
  • the cargo/VEPEP-5 molar ratio depends on the nature and size of the cargo, but is generally comprised between 1/1 and 1/50.
  • the cargo/VEPEP-5 molar ratio preferably ranges from 1/5 to 1 /20.
  • the cargo VEPEP-5 molar ratio preferably ranges from 1/3 to 1/10.
  • the cargo/VEPEP-5 molar ratio preferably ranges from 1/10 to 1 /20
  • the VEPEP-5 peptides comprise a polyethylene glycol group or an acetyl group covalently linked to their N-terminus, and/or a cysteamide group covalently linked to their C-terminus.
  • the above complexes can be advantageously used as "core shells" for obtaining bigger complexes, or nanoparticles, by an additional step of coating the cargo/VEPEP-5 complex with another layer of cell -penetrating peptides, which can be identical to or different from the VEPEP-5 peptides described above.
  • VEPEP-5/CADY wherein CADY is a CPP as described in EP 1795539 and in [1 1]
  • VEPEP-5/PEP-1 wherein Pep-1 is a CPP as described in [8]
  • VEPEP- 5/MPG wherein MPG is a CPP as described in US7.514,530 and in [7, 10]
  • nanoparticles with an outer layer made of a CPP belonging to another VEPEP family for example selected from the following list:
  • VEPEP-3a Ac-XiKWFERWFREWPRKR -cysteamide (SEQ ID No: 1 1)
  • VEPEP-3b Ac-X , KWWERWWRE PRKRK-cy steamide (SEQ ID No: 12)
  • VEPEP-3d Ac-X 1 RW YE W YTE FP RRRR-cysteam i de (SEQ ID No: 14), - VEPEP-3e: Ac-X , RWWRLWWRS WFRLWRR-cysteamide (SEQ ID No: 15)
  • VEPEP-6d Ac- i L WRALWRL WRSL WRLWRKA-cysteamide (SEQ ID No: 22)
  • VEPEP-9b2 Ac-X , R WWLRWASR WFLS WRWWR-cysteamide (SEQ ID No: 28)
  • VEPEP-9c2 Ac-X, RWWLRWASRWAPS WRWWR-cysteamide (SEQ ID No: 30)
  • - ST-VEPEP-6c Ac-X, LFAR S LWRLLRS S LWRLL WK-cysteamide (SEQ ID No: 44), as well as variants thereof (regarding the amino acid sequence and/or the N- and C- terminal chemical groups), wherein X] is beta-A or S and wherein the residues followed by an inferior "s" are linked by a hydrocarbon linkage.
  • Preferred variants of the above sequences for forming nanoparticles according to the invention are PEGylated at their N- terminal extremity instead of being acetylated.
  • Nanoparticles made of a "core shell” comprising a cargo and a first carrier molecule, surrounded by VEPEP-5 peptides. These are herein referred to as " ⁇ -5" particles.
  • NA OPEP-5 technology constitutes a "custom-built” delivery system containing a common core particle, trapping therapeutic molecule, with surface VEPEP-5 peptides which are preferably functionalized for tumour or tissue targeting in vivo. From a structural point of view, NANOPEP-5 particles are constituted by a "core” which is coated by a layer of VEPEP-5 peptides.
  • the "core” corresponds to a complex comprising a cargo and a vector or carrier such as a first cell-penetrating peptide, a liposome, a polycationic structure, a carbon nanoparticle, etc.
  • a vector or carrier such as a first cell-penetrating peptide, a liposome, a polycationic structure, a carbon nanoparticle, etc.
  • the layer of VEPEP-5 peptides peripheral peptide
  • Functionalizing NANOPEP-5 particle surface with either cholesterol, lipid, PEG-molecules improves particles stability in vivo, favours their administration by either systemic or topical route and allows rapid liberation of active cargoes within tumor cells or tissues.
  • NANOPEP-5 particles Functionalization of the surface of NANOPEP-5 particles with small FAB fragments, peptides, antibodies and lipids has been shown to favour in vivo tissue or tumor targeting. Also, Functionalizing NANOPEP-5 particle surface with polysaccharide such as PLGA, can be used as formulation for slow release of drug and cargo and allow a long term response in vivo. N-terminal PEGylation o at least part of the VEPEP-5 peptides surrounding the NANOPEP-5 particles increases the biodistribution of cargoes in the tumour, probably by stabilizing the NANOPEP-5 particles in the plasma.
  • NANOPEP-5 technology improves both cellular and in vivo delivery of biologically active cargoes and has been validated on a large set of cell lines including adherent and suspension cell lines, hard to transfect cell lines. NANOPEP-5 particles strongly interact with cell membranes and enter the cell independently of the endosomal pathway or rapidly escape from early endosomes. NANOPEP-5 technology presents several advantages including rapid delivery with very high efficiency, stability in physiological buffers, protection of the cargo against degradation, lack of toxicity and of sensitivity to serum, ability of forming mix nanoparticlcs, can be functionalized can be applied to the delivery of different types of cargoes into a large variety of cell lines as well as in animal models, thereby constituting powerful tools for basic research and therapeutic applications. NANOPEP-5 technology can be applied both at therapeutic and d iagno sti c/theragnostic levels, as well as for imaging.
  • the cargo is complexed to a first cell-penetrating peptide, which can be, for example, selected amongst CADY, MPG, PEP-1 , PPTG1 , poly Arginine motif, VEPEP-familly peptide (VEPEP-3, VEPEP-5, VEPEP-6, VEPEP-9, stapled or not) as described above (such as SEQ ID Nos: 1 to 7 and 11 to 44 and variants thereof), or any other known CPP.
  • This cargo/CPP complex is then coated with a layer of VEPEP-5 peptides.
  • the skilled artisan will advantageously choose the first CPP depending on the nature of the cargo, so that the complex of cargo and first CPP is stable. Hence, a wide diversity of cargoes can be included in NANOPEP-5 particles.
  • the corc/VEPEP-5 molar ratio depends on the nature and size of the core, but is generally comprised between 1/1 and 1/50,
  • the core/peripheral VEPEP-5 molar ratio preferably ranges from 1/5 to 1/30, depending on the nature of peptide cargo (hydrophobicity and charge).
  • the size of the nanoparticle is between 20 and 300 nm.
  • the VEPEP-5 peptides forming the peripheral layer of the nanoparticles comprise a poly-ethylene glycol or an acetyl group covalently linked to their N-terminus. and/or a cysteamide group covalently linked to their C-terminus.
  • the core shell of the particles is coated with a VEPEP-5 peptide functional! zed with NTA (for example, a VEPEP-5 peptide with nitrilotriacetic acid covalently linked to its C-terminus).
  • NTA for example, a VEPEP-5 peptide with nitrilotriacetic acid covalently linked to its C-terminus.
  • At least part of the VEPEP-5 cell-penetrating peptides are bound to a targeting molecule.
  • a targeting molecule include antibodies, nanobodies and Fc or FAB fragments (for example targeting HEK2/MUC 1 /EGF/XCCR4).
  • Non-limitative examples of such ligands and homing peptides are: RGD-peptide, homing targeting peptides (brain NTl peptide, Ganglion GM 1 peptide, as well as all other previously described peptides for tissues and cell line targeting), folic acid, polysaccharides, and matrix metalloprotease targeting peptide motif (MMP-9 or MMP3 for tumour selectivity).
  • the complexes or nanoparticles are formulated se that they can be stored during several months without losing their stability and functional efficacy.
  • the complexes and nanoparticles of the invention ca advantageously be Iyophilized in the presence of a sugar.
  • sugars which can be used to that aim are sucrose, glucose, manitol and a mix thereof, and they can be used, for example, in a concentration ranging from 5% to 20%, preferably 5% to 10%, it being understood that a concentration of 5% is obtained by adding 5 grams per litre of solution before lyophilization.
  • Another aspect of the present invention is the use of a complex or nanoparticle as above-described, as a medicament and as a marker or an imaging agent.
  • the present invention also pertains to a therapeutic, cosmetic or diagnostic composition
  • a therapeutic, cosmetic or diagnostic composition comprising a complex or a nanoparticle as described above.
  • a composition comprising a complex or nanoparticle having a peptide targeting protein protein interactions, involving essential protein CD and Cyclin required for cell cycle progression as a cargo, and a targeting molecule specific for tumour cells (for example: RGD-peptide, folic acid, MUC-1 or HEK2 antibodies or nanobodies ), is part of the present invention.
  • this composition can be formulated for intravenous, intratumoral, topical, intrarectal, intranasal, transdermal, or intradermal administration, or for administration via a mouth spray, or for administration as a subcutaneous implant for slo w release of a drug.
  • the present invention also pertains to a method for delivering a molecule into a cell in vitro, comprising a step of putting said cell into contact with a complex or nanoparticle as described above.
  • Short peptides do not induce allergic response and therefore can be administered by trans-dermal route and topically without any risk of inflammatory response, in contrast to longer peptides. Moreover, the risk of immune response following intravenous injection of VEPEP-5 is negligible.
  • All peptides were synthesized by solid-phase peptide synthesis using AEDI-expensin resin with (fluorenylmethoxy)-carbonyl (Fmoc) on a Pioneer Peptide Synthesizer (Pioneer TM, Applied Biosy stems, Foster City, CA) starting from Fmoc-PAL- PEG-PS resin at a 0.2 mmol scale.
  • the coupling reactions were performed with 0.5 M of HATU in the presence of 1 M of DIEA.
  • Protecting group removal and final cleavage from the resin were carried out with TFA/Phenol/H ⁇ O/Thioanisol/Ethanedithiol (82.5/5/5/5/2.5 %) for 3 h 30 mill.
  • X Cholesterol, Pegylation, stearyl, palmitoyl, small FC or FAB fragments, nanobody.
  • nitrilotriacetic acid (2 x NTA) tissues targeting peptides (brain, lung, lymphe node, pancreas).
  • VEPEP-5 peptides are tryptophan rich peptides: they are highly versatile and show a strong structural polymorphism. VEPEP-5 are unfolded in solution in free form as well as in the presence of lipid or artificial cellular membranes or of cargo s such as peptide or small molecules.
  • Short oligonucleotides PNA and 5' Alexa 700 or cy5 fluorescently labelled PNA were synthesized by Eurogentec (Belgium) according to the following sequences.
  • Mean particle size distribution was determined with a Coulter N4 Plus (Coulter-Beckman) at 25 °C for 3 min per measurement and zeta potential was measured with Zetasizer 4 apparatus (Malvern Ltd).
  • Adherent HS68 fibroblasts HeLa. PC3, Jurkat. CEM-SS and U20S MCF-7 cell lines (from American Type Culture Collection (ATCC)), as well as MEF and PBMC were cultured in Dulbecco's Modified Eagle's Medium supplemented with 2mM glutamine, 1% antibiotics (streptomycin 10,000 ⁇ / ⁇ 1, penicillin, 10,000 IU/ ml) and 10% (w/v) foetal calf serum (FCS), at 37°C in a humidified atmosphere containing 5% C ( Stock solutions of VEPEP-5/peptide particles were prepared by complexing 1 ⁇ peptide with VEPEP-5 peptides at a molar ratio of 1/20 for 30 min at 37°C.
  • VEPEP-5 -carrier /peptide from 500 nM to 1 ⁇ were obtained by serial dilution of the stock complexes in PBS, in order to preserve the same VEPEP-5-carrier/peptide ratio.
  • FCS foetal calf serum
  • VEPEP-5/peptide or VEPEP-5/SHM complexes Toxicity of VEPEP-5/peptide or VEPEP-5/SHM complexes was investigated on Hela and HS-68 cell lines. 30,000 cells seeded in 24- well plated the day prior transfection. were incubated with increasing concentrations of peptide or SHM complexed with VEPEP-5 at a 20/1 molar ratio ranging from 1 to 5 ⁇ , for 30 min prior to addition of medium to reach a final 10% concentration of FCS. Cytotoxic response was measured 24hr later by colorimetric MTT assay (Sigma, Germany), respectively. For MTT assay, cell culture medium was removed and replaced with PBS containing 2.5 mg/ml of MTT for 4hr. Results correspond to the average of 3 separate experiments.
  • Example 2 VEPEP-5 peptides form stable nanostructures with different cargoes
  • VEPEP-5 peptides (Short-6 (SEQ ID No: 1), Short-7 (SEQ ID No: 2) and Short 1 1 (SEQ ID No: 6) form stable complexes with peptides, cyclic peptide, PNA and Protein.
  • the binding of cargos was monitored by fluorescence spectroscopy using the both intrinsic Trp group of VEPEP-5 (3-4 Trp-residues) and extrinsic fltiorescently labelled cargoes (using Cy5 or FITC). Curve fitting revealed that VEPEP-5 strongly binds the different cargoes with dissociation constant in the nanomolar range (Table 1 and Figure 1 ).
  • Cyclic peptide (PC4), PNA and protein ( 15 kDa).
  • VEPEP-5 peptides also form stable particles with small aromatic molecules including doxorubicin, porphyrin and charges molecules including nucleotide, nucleoside and peptide-analog of nucleic acids or fluorescent dyes (Figure 1).
  • the dissociation constant for small hydrophobic molecule ranges between 0.05 to 0.1 ⁇ , depending on the nature of the dyes and of the peptides (Table 2).
  • VEPEP-5/Cargo complexes characterization VEPEP-5/Cargo complexes characterization.
  • SHM small hydrophobic molecules (porphyrin, FAM-G, doxorubicin)
  • the size of the particles was monitored by dynamic light scattering.
  • the optimal VEPEP-5 peptide/cargo (peptide and SUM) molar ratio is ranging between 1/10 to 1/30 ( Figure 2).
  • the size of the particles is of about 50 to 200 nanometer in diameter.
  • Example 3 VEPEP-5 mediated delivery of peptide and cyclic peptide in different cell lines
  • VEPEP-5 peptides (Short-6. Short-7 and Short 1 1) have been used for the delivery of different peptides into different cell lines, including primary cell lines, stem cell lines and challenging cell lines. Peptide delivery was monitored using three approaches, fluorescence spectroscopy and monitoring biological responses (anti proliferation)
  • VEPEPS-mediated delivery of peptide targeting Cdk2/cyclin A blocks cancer cell proliferation
  • VEPEP-5 Short-6, Short-7 and Short 1 1
  • mediated delivery of C4 peptide induced a robust biological response associated with specific cell cycle arrest ( Figure 3).
  • a peptide C4 concentration of 100 iiM was sufficient to block proliferation of Hela and MCF7 cells.
  • Short6 peptide an IC50 of 80 ⁇ 20 nM and 50 ⁇ 10 nM were estimated for C4 peptides respectively o Hela and MCF7.
  • short7 peptide an IC50 of 45 ⁇ 10 nM and 80 ⁇ 20 nM were estimated for C4 peptides respectively on Hela and MCF7.
  • VEPEP-5 mediated delivery of PNA molecule in different cell lines
  • VEPEP-5 Short-6, Short-7 and Short 1 1
  • PNA and morpholino nucleic acid analog
  • Uptake was monitored by following biological response (Cyclin B l knockdown).
  • Short-6, Short-7 and Short 1 1 for the delivery of PNA antisense targeting Cyclin B l as previously described by Morris et al. [ 13].
  • VEPEP-5 mediated delivery of small hydrophobic molecules in different cell lines
  • VEPEP-5 peptides (Short-6, Short-7 and Short 1 1 ) have been used for the delivery of different small hydrophobic molecules including doxorubicin and porphyrin on different cell lines including primary cell lines and challenging cell lines. VEPEP-5 peptides form stable particles with small aromatic molecules (table 2). Effect of VEPEP-5 mediated delivery of doxorubicin and porphyrin have been investigated on cancer cell viability. Dose-response experiments performed on cultured cells revealed that VEPEP-5 mediated delivery of doxorubicin and porphyrin induced a biological response associated to cell cycle arrest and decrease in viability o MCF7, Hela cancer cells ( Figure 5).
  • VEPEP-5 mediated delivery of proteins in different cell lines
  • VEPEP-5 have been used for the delivery of different proteins into different cell lines, including primary cell lines, stem cell lines and challenging cell lines. Protein uptake was monitored using fluorescence spectroscopy and FACS analysis. GFP/RFP or Fluorescent labelled proteins were visualized in the different cell lines using fluorescence microscopy or FACS sorting (Table 5). in most of the cell lines, the uptake of RFP; GFP, Cy-5 labelled proteins is more than 50% of the cells.
  • Example 4 VEPEPS-mediated delivery of peptide/P A and SHM is not toxic.

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Abstract

L'invention concerne un peptide pénétrant dans les cellules, caractérisé en ce qu'il comprend une séquence d'acides aminés consistant en une bêta-alanine ou une sérine en position N-terminale, liée à une séquence constituée de 9 acides aminés consécutifs de la séquence RXWXRLWXRLR (SEQ ID NO: 7), dans laquelle X en position 2 représente R ou S et X en positions 4 et 8 représentent, indépendamment l'un de l'autre, W ou F.
EP13773244.2A 2012-10-04 2013-10-04 Peptides pénétrant dans les cellules pour la livraison intracellulaire de molécules Not-in-force EP2928907B1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022020782A1 (fr) 2020-07-24 2022-01-27 Aadigen, Llc Compositions et méthodes de traitement d'infections virales

Families Citing this family (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8822663B2 (en) 2010-08-06 2014-09-02 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
CA3162352A1 (fr) 2010-10-01 2012-04-05 Modernatx, Inc. Nucleosides, nucleotides et acides nucleiques modifies, et utilisations connexes
AU2012236099A1 (en) 2011-03-31 2013-10-03 Moderna Therapeutics, Inc. Delivery and formulation of engineered nucleic acids
WO2012137036A1 (fr) 2011-04-04 2012-10-11 Centre National De La Recherche Scientifique Peptides capables de pénétrer dans des cellules pour administration intracellulaire de molécules
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
EP2791160B1 (fr) 2011-12-16 2022-03-02 ModernaTX, Inc. Compositions de mrna modifiés
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
AU2013243946A1 (en) 2012-04-02 2014-10-30 Moderna Therapeutics, Inc. Modified polynucleotides for the production of membrane proteins
US9303079B2 (en) 2012-04-02 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
WO2014053881A1 (fr) 2012-10-04 2014-04-10 Centre National De La Recherche Scientifique Peptides pénétrant dans les cellules pour l'administration intracellulaire de molécules
WO2014053879A1 (fr) 2012-10-04 2014-04-10 Centre National De La Recherche Scientifique Peptides pénétrant dans les cellules pour l'administration intracellulaire de molécules
WO2014053880A1 (fr) 2012-10-04 2014-04-10 Centre National De La Recherche Scientifique Peptides pénétrant dans les cellules pour l'administration intracellulaire de molécules
LT2922554T (lt) 2012-11-26 2022-06-27 Modernatx, Inc. Terminaliai modifikuota rnr
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
CA2926218A1 (fr) 2013-10-03 2015-04-09 Moderna Therapeutics, Inc. Polynucleotides codant pour un recepteur de lipoproteines de faible densite
US10815276B2 (en) 2014-05-21 2020-10-27 Entrada Therapeutics, Inc. Cell penetrating peptides and methods of making and using thereof
ES2739613T3 (es) 2014-05-21 2020-02-03 Entrada Therapeutics Inc Péptidos que penetran en las células y sus métodos de preparación y su uso
EP2977059A1 (fr) 2014-07-22 2016-01-27 Vaxon Biotech Polypeptide immunogène composé de peptides cryptiques optimisés dérivés d'antigènes tumoraux restreints hla-b7 et leurs utilisations
US10456443B2 (en) 2014-08-27 2019-10-29 Ohio State Innovation Foundation Peptidyl calcineurin inhibitors
US10745440B2 (en) 2014-12-24 2020-08-18 Aadigen, Llc Peptides and nanoparticles for intracellular delivery of molecules
CN105722026A (zh) * 2016-01-27 2016-06-29 东华大学 监狱犯人定位跟踪系统
CA3025523A1 (fr) 2016-05-27 2017-11-30 Aadigen, Llc Peptides et nanoparticules pour l'administration intracellulaire de molecules d'edition du genome
JP2019537600A (ja) 2016-11-02 2019-12-26 ナノプロティアジェン， リミテッド ポリマーナノ粒子
CA3043464A1 (fr) 2016-11-09 2018-05-17 Ohio State Innovation Foundation Peptides de penetration cellulaire a liaisons disulfure, leurs procedes de preparation et d'utilisation
TW201825510A (zh) * 2016-11-22 2018-07-16 俄亥俄州立創新基金會 細胞穿透肽序列
US10913773B2 (en) 2016-11-22 2021-02-09 Ohio State Innovation Foundation Bicyclic peptidyl inhibitor of tumor necrosis factor-alpha
WO2018098282A2 (fr) 2016-11-22 2018-05-31 Ohio State Innovation Foundation Peptides cycliques pénétrant dans les cellules comprenant des motifs en épingle à cheveux et leurs procédés de fabrication et d'utilisation
WO2018102317A1 (fr) * 2016-11-29 2018-06-07 The Regents Of The University Of California Modulation de p53 pour le traitement du cancer
CN110997693A (zh) * 2017-06-07 2020-04-10 阿德克斯公司 τ聚集抑制剂
US11786607B2 (en) 2017-06-15 2023-10-17 Modernatx, Inc. RNA formulations
WO2019046809A1 (fr) 2017-08-31 2019-03-07 Modernatx, Inc. Procédés de fabrication de nanoparticules lipidiques
WO2019148194A2 (fr) 2018-01-29 2019-08-01 Ohio State Innovation Foundation Inhibiteurs peptidyliques de l'interaction calcineurine-nfat
EP3755351A4 (fr) 2018-02-22 2021-11-24 Entrada Therapeutics, Inc. Compositions et méthodes de traitement de l'encéphalopathie mitochondriale neurogastro-intestinale
EP3790890A4 (fr) 2018-05-09 2022-03-02 Ohio State Innovation Foundation Peptides cycliques de pénétration cellulaire avec un ou plusieurs résidus hydrophobes
CN110790821B (zh) * 2019-11-19 2022-09-02 南阳师范学院 一种细胞核穿透肽及其用途

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7514530B2 (en) 2004-04-26 2009-04-07 Centre National De La Recherche Scientifique Peptide carrier for delivering siRNA into mammalian cells
US8242081B2 (en) * 2005-12-06 2012-08-14 Centre National De La Recherche Scientifique Cell penetrating peptides for intracellular delivery of molecules
DE602005025141D1 (de) 2005-12-06 2011-01-13 Centre Nat Rech Scient In Zellen eindringende Peptide als Träger für Moleküle
US7906484B2 (en) 2006-09-21 2011-03-15 Alnylam Pharmaceuticals, Inc. Complex for transferring an anionic substance into a cell
CA3102008A1 (fr) 2010-06-02 2011-12-08 Alnylam Pharmaceuticals, Inc. Compositions et methodes pour traiter une fibrose hepatique
WO2012137036A1 (fr) 2011-04-04 2012-10-11 Centre National De La Recherche Scientifique Peptides capables de pénétrer dans des cellules pour administration intracellulaire de molécules
CA2873745A1 (fr) 2012-05-16 2013-11-21 Aadigen, Llc Modulation multi-cibles pour traiter la fibrose et des affections inflammatoires
WO2014053881A1 (fr) 2012-10-04 2014-04-10 Centre National De La Recherche Scientifique Peptides pénétrant dans les cellules pour l'administration intracellulaire de molécules
WO2014053880A1 (fr) 2012-10-04 2014-04-10 Centre National De La Recherche Scientifique Peptides pénétrant dans les cellules pour l'administration intracellulaire de molécules
WO2014053879A1 (fr) 2012-10-04 2014-04-10 Centre National De La Recherche Scientifique Peptides pénétrant dans les cellules pour l'administration intracellulaire de molécules

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2014053629A1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022020782A1 (fr) 2020-07-24 2022-01-27 Aadigen, Llc Compositions et méthodes de traitement d'infections virales

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US10118944B2 (en) 2018-11-06

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